In the present study, volatile compound analysis of olive oil samples belonging to ten Greek cultivars was carried out. A total of 167 olive oil samples collected from two consecutive harvest years were analyzed by Head Space-Solid Phase Microextraction-Gas Chromatography/Mass Spectrometry (HS-SPME-GC/MS). Volatile compound data were combined with chemometric methods  and Linear Discriminant Analysis (LDA)) with the aim not only to differentiate olive oils but also to identify characteristic volatile compounds that would enable differentiation of botanical origin (marker compounds). The application of Stepwise LDA (SLDA) effectively reduced the large number of statistically significant volatile compounds involved in the differentiation process, and thus, led to a set of parameters, the majority of which belong to compounds that are highly dependent on variety. In addition, the use of these marker compounds resulted in an increased correct classification rate (85.6%) using the cross-validation method indicating the validity of the model developed despite the use of a large number of dependent variables (cultivars). Olive tree cultivation, one of the oldest and most important agricultural activities, has led to the diversification of olives into a large number of cultivars. The olive fruit cultivar is a major determinant of olive oil quality due to differences in specific aroma and taste, phenolic content, etc. leading to a variety of olive oils, each with unique flavor characteristics and stability. In Greece more than 30 cultivars of olives exist, and most are characteristic of cultivation area  ,2.According to the International Food Authenticity Assurance Organization (ΙFAAO) , “food aSophisticated analytical techniques used for the authentication of olive oil include: analysis of volatile compounds by the HS-SPME-GC/MS method ,2, protoOlive oil is generally defined on the basis of its sensory as well as its physico-chemical characteristics. Within sensory evaluation, many volatile compounds are considered responsible for desirable and undesirable odor attributes of olive oil. As volatiles are influenced by many factors  , variatiThe sensory evaluation protocol for olive oil, namely, the Panel Test, has been established by the International Olive Oil Council  and EU rper se or in combination with other analytical parameters resulted in a greater aggregation of olive oil samples in respective cultivars. This fact and the potential of this analytical technique have been used in the present study, the aim of which was to determine those volatiles which have a higher discriminant ability for cultivar differentiation of olive oil. For this purpose, the volatile compound data from ten different olive oil cultivars were combined and analyzed using Multivariate Analysis of Variance (MANOVA) and Linear Discriminant Analysis (LDA). As the number of significant variables (p < 0.05) that derive from MANOVA may be quite large, stepwise LDA (SLDA) was then applied in order to reduce this number of volatiles to that which could be considered as the best set of predictors-markers of authenticity in relation to the herein studied cultivars. Previously published data on the differentiation of olive oil cultivars by means of instrumental analysis combined with chemometrics have led to the conclusion that this combination is a very powerful tool for the determination of authenticity of olive oil ,2,25. LiLadolia Kerkyras, Galano, Adramitiani, Samothraki, Athinolia, Hontrolia, Koutsourelia, Kolovi, Topiki Makris and Manaki ; results were expressed as μg/kg. All determinations were carried out in triplicate.The determination of volatile compounds was carried out according Kosma et al. , using SStatistical treatment of data was performed using SPSS 25.0 software. Data were subjected to MANOVA in order to determine those variables that are significant for the differentiation of olive oil cultivar. Cultivar was taken as the independent variable, while volatile compounds were taken as the dependent variables. Pillai’s Trace and Wilks’ Lambda indices were computed to determine a possible significant effect of experimental parameter values on olive oil cultivar. LDA was then applied using the selected dependent variables in order to explore the potential for classification of olive oil samples according to cultivar. Original and leave-one-out cross-validation methods were used to test the prediction classification ability. In the original method, the prediction rate results from the contribution of all cases in the discriminant functions while in cross-validation, a randomly chosen parameter, is classified in a group based on a discriminant function, created by all the other parameters (except the randomly chosen one). This procedure is repeated for all the parameters of the tested sample. The homogeneity of variability was tested by application of the Box M index . Since the number of volatile compounds resulting from the analysis is quite large, SLDA was used as a final step in order to determine those variables that show higher discriminant ability. The SLDA method is based on the creation of an initial model which does not include any of the significant variables-predictors. The predictors are introduced in the analysis sequentially, one-at-a-time, until all are included in the model. The SLDA classification method applies a forward variable selection algorithm using Wilks’ Lambda as a selection criterion and the F-statistical factor in order to determine the significance of changes in Wilks’ Lambda when the impact of a new variable is evaluated . Before 232 and K270) did not exceed the internationally established limits set by the EU Regulation [Galano samples to 1.8% ± 1.6 in Adramitiani samples which along with samples from Ladolia Kerkyras, Samothraki and Athinolia  recorded higher acidity values and were categorized as virgin olive oil. Furthermore, Ladolia Kerkyras recorded the highest K232 value (2.76 ± 1.02) this categorizing this oil as “lampante” while the other samples remained lower than the internationally established limit of 2.50 [As shown in gulation . Specifi of 2.50 . Lanolia Kerkyras were also recorded. These samples were also categorized as “lampante” and according to the authors this may be related to the method of olive fruit collection, where in certain olive orchards in Kerkyra, fruits are left to fall off the olive tree and are collected in nets on the ground. In general, this collecting method is not a common practice as it causes damages to the fruit.In a similar study by Pouliarekou et al.  who clasLadolia Kerkyras , Topiki Makris  and Hontrolia .Sixty volatile compounds were identified and semi-quantified in olive oil samples tested . These v(E)-2-Hexenal was the most abundant aldehyde identified in all samples tested, related to olive fruit maturity (characteristic of olive cultivar) and oxidation stage of olive oil [Topiki Makris cultivar . Hexanal followed, recording its highest concentration in the Ladolia Kerkyras (3774.7 ± 2983.8 μg/kg) and Topiki Makris (3189.8 ± 1057.7 μg/kg) samples. It should be noted that the relatively high hexanal content observed in the olive oil samples tested does not necessarily indicate either oxidized olive oils or olive oils in the early stages of oxidation. According to Morales et al. [E)-2-Pentenal derives from the lipoxygenase pathway through the action of alkoxy radicals on linolenic acid 13-hydroperoxides producing the corresponding alcohol ((Z)-2-pentenol) which is subsequently oxidized. (E)-2-pentenal was not detected in the Adramitiani, Samothrakis, Athinolia, Ladolia Kerkyras and Manaki cultivars, while it showed the highest concentration in the Topiki Makris cultivar (67.7 ± 55.5 μg/kg). According to Morales et al. [Manaki cultivar. The lipoxygenase pathway has a major contribution to virgin olive oil aroma as a wide variety of volatile compounds are produced through this biological pathway . Aldehydlive oil ,35 recor(E)-2-Hexenol was the most abundant alcohol recording its highest concentration in the Koutsourelia (1397.7 ± 1385.0 μg/kg), while hexanol, the second most abundant alcohol recorded the highest concentration in the Kolovi (1704.6 ± 1079.5 μg/kg) samples. These two alcohols can be used for cultivar differentiation while (E)-2-hexenol is responsible for the characteristic “green” aroma notes of the olive oil; hexanol’s odor perception is considered as fruity, banana like and grassy [Samothrakis samples to 165.5 ± 125.4 μg/kg in Koutsourelia.Aldehydes are reduced to alcohols through the action of alcohol dehydrogenase. d grassy , was preKolovi (3307.1 μg/kg), Manaki (2870.6 μg/kg) and Koutsourelia (2710.4 μg/kg) samples recorded the highest total concentrations compared to the other cultivars while esters were not identified in the volatile fraction of the Galano and Hontrolia olive oils. The absence of esters in the volatile fraction of certain cultivars may be due to the action of the enzyme alcohol acyltransferase. The activity of this enzyme is significantly influenced by pH (6.8–8) and temperature (35 °C), as well as the availability of the appropriate substrate. The activity of alcohol acyltransferase can be enhanced by cultivar selection as well as by modifying olive oil extraction conditions, i.e., operation at lower temperatures to prevent inactivation of the enzymes and promotion of esterification reactions [Another important chemical class that has a major contribution to olive oil aroma is that of esters. Esters derive from the lipoxygenase pathway, through the action of alcohol acyltransferase that catalyzes the formation of acetate esters through acetyl-CoA derivatives. Despite the fact that esters comprise minor components of olive oil aroma their contribution is quite significant as they complement aroma with sweet and pleasant notes ,39. KoloHontrolia (3630.0 μg/kg) and Ladolia Kerkyras (3570.2 μg/kg) while Samothrakis recorded the lowest concentration (206.0 μg/kg). 1-Penten-3-one also derives from the lipoxygenase pathway and is positively correlated to bitter taste [Samothrakis and Topiki Makris cultivars . 2-Pentanone and 3-pentanone resulting from homolytic cleavage processes, are also responsible for the green notes of aroma [Kolovi cultivar . Furthermore, 6-methyl-5-hepten-2-one was present in all samples tested with the exception of the Manaki samples. This ketone is produced through the action of the pseudomonads that break down terpene alcohols present in olive oil [Adramitiani cultivar (260.6 ± 240.3 μg/kg). Regarding ketones, their highest total concentration was recorded in the er taste . In olivof aroma . The higlive oil . The 6-MLadolia Kerkyras (13006.4 μg/kg) and Hondrolia (15966.5 μg/kg). The lowest total amount of hydrocarbons was recorded in the Kolovi cultivar (1351.1 μg/kg). 3-Ethyl-1,5-octadiene deriving from the lipoxygenase pathway, recorded significant variations in all samples tested, indicating the influence of cultivar and/or environmental conditions on olive tree growing. Specifically, the highest value of 3-ethyl-1,5-octadiene was recorded in the Topiki Makris (858.9 ± 343.2 μg/kg) and the lowest in Athinolia (198.7 ± 148.4 μg/kg).In the intact tissues of the olive fruit there are only a few volatile components, mainly hydrocarbons which do not contribute to fruit aroma. Most of the volatile compounds form as soon as the tissues are damaged and the various enzymatic activities, previously mentioned, initiate . SignifiLadolia Kerkyras (3781.4 μg/kg), Adramitiani (5476.3 μg/kg) and Kolovi (4405.3 μg/kg). The terpenes identified and semi-quantified were α-pinene, δ-3-carene, p-cymene, dl-limonene, (E)-β-ocimene, allo-ocimene, α-copaene, (E)-β-farnesene, -α-farnesene and α-muurolene.According to Bubola et al. , some vop = 0.001 < 0.05) and Wilks’ Lambda = 0.001  index values showed the existence of a significant multivariable effect of cultivar on the identity of volatile compounds.As a first step, the 167 olive oil samples were subjected to MANOVA in order to determine those volatile compounds which are significant for cultivar differentiation. Dependent variables included the total 60 volatile compounds identified and semi-quantified while cultivar was taken as the independent variable. Pillai’s Trace = 7.049  for cultivar differentiation and thus, were subjected to LDA, a second step of the analysis. Results showed that three statistically significant discriminant functions are formed (p = 0.050) was insignificant at the 95% confidence level indicating the existence of uniformity of sample variability for each cultivar. In Galano are very well differentiated from the other cultivars. In Samothraki, Topiki Makris and Adramitiani cultivars are well differentiated while the other cultivars are overlapping. The overall correct classification rate was 99.4% for the original and 83.2% for the cross-validation method. Correct cultivar classification (100%) was achieved for Adramitiani, Samothraki and Galano cultivars. Fifty-five volatiles were found to be significant  previously published work [Galano and Samothraki cultivars were fully differentiated from the rest of the cultivars investigated. Furthermore, regarding the other five cultivars  the classification rate achieved in previous work was 100% for the original and 82.4% for the cross-validation method also leading to very satisfactory differentiation of the tested cultivars [Galano, Samothrakis, Topiki Makris and Adramitiani) were well differentiated.Regarding five of the above olive cultivars  was achieved only for Galano, Adramitiani and Topiki Makris cultivars. As the final step of data statistical treatment, SLDA was used in order to select the variables with the higher discriminant ability. Of the 55 significant volatile compounds only 17 were found to have a higher discriminant ability . Three s(E)-2-hexen-1-ol, (E)-2-pentenal, (E)-2-hexenal and hexyl acetate. This fact indicates strong dependence of this biological pathway on cultivar as these compounds have a major contribution to virgin olive oil aroma with a wide variety of volatile compounds being produced through this biological pathway [E)-2-hexenal, represents an effective tool for differentiating single-variety oils from different varieties. Focusing on specific volatile compound-markers, Kesen et al. [E)-2-hexenal, followed by hexanal, in Turkish olive oil samples belonging to the Halhali cultivar. Furthermore, Bubola et al. [E)-2-hexenal was the most abundant aldehyde, while Tanouti et al. [E)-hex-2-enal, Z-3-hexen-1-ol and 1-hexanol, as in the present study. Finally, Issaoui et al. [E)-2-hexenal and 1-hexanol can be used as potential indicators, in this case for geographical differentiation. Ethanol, according to Kalua et al. [E)-β-ocimene and α-copaene) were identified as volatile markers. According to Zunin et al. [Of the 17 volatile compound-markers, 5 derive through the lipoxygenase pathway [1-penten-3-ol,  pathway . Pizarro pathway , in an a pathway  the effen et al.  reporteda et al.  reportedi et al.  recordedi et al.  who studa et al. , resultsa et al. ,38,41. Ta et al. ,44. In tn et al. , α-copaeE)-2-hexenal, (E)-2-hexenol, 1-penten-3-ol, (E)-2-pentenal) while three are terpenes -β-ocimene, dl-limonene), indicating a strong dependence of cultivar on the formation of olive oil’s volatile fraction. The resulting distribution diagram showed that the cultivars Galano, Samothrakis, Topiki Makris and Adramitiani were clearly differentiated. The results are quite encouraging, demonstrating the validity of the statistical model developed for the authentication of olive oil in relation to the differentiation of olive cultivars.In the present study, volatile compound analysis from olive oil samples belonging to ten different cultivars from Greece was carried out in an effort to (i) to differentiate cultivar based on volatile compounds and (ii) to investigate the selection of potential markers leading to a successful cultivar differentiation. The results of the statistical treatment (MANOVA/LDA) showed that the differentiation of olive oil samples according to cultivar is possible despite the quite large number of cultivars investigated . Furthermore, the results obtained after the application of SLDA to the selected set of variables (55 volatile compounds) led to a quite reduced set of data (17 volatile compounds). These compounds provide a higher discriminant ability compared to the other volatiles, increasing the classification rate to 85.6% with the application of the cross-validation method. The specific volatile compounds identified as markers included a total of seventeen compounds. Of these, five derive from the lipoxygenase pathway (hexyl acetate, (


Well-ordered bioreceptors on atomically flat Au surfaces can be a high-performance biosensor. Cardiac troponin I proteins (cTnIs) have been regarded as a specific biomarker for acute myocardial infarction (AMI). Here, we report the accurate detection of cTnIs using an aptamer-immobilized Au nanoplate platform. The single-crystalline and atomically flat Au nanoplate was characterized by atomic force microscopy. For the precise detection of cTnI, we immobilized an aptamer that can strongly bind to cTnI onto an atomically flat Au nanoplate. Using the aptamer-immobilized Au nanoplate, cTnIs were successfully detected at a concentration of 100 aM (2.4 fg/mL) in buffer solution. Furthermore, cTnIs in serum could be identified at a concentration of 100 fM (2.4 pg/mL). The total assay time was ~7 h. Importantly, the aptamer-immobilized Au nanoplate enabled us to diagnose AMI patients accurately, suggesting the potential of the present method in the diagnosis of AMI. Acute myocardial infarction (AMI) is a common cause of death globally . AccordiAtomically flat surfaced nanomaterials have been interfaced with various kinds of systems, improving nano-devices remarkably ,21,22,23Herein, we demonstrate that the optimal immobilization of an aptamer onto the atomically flat Au nanoplates can be a high-performance biosensor for cTnI. The atomic force microscopy (AFM) results show that the aptamer is well immobilized on the Au nanoplate and also provides evidence that the cTnIs can be captured on the aptamer-immobilized Au nanoplate. In addition, aptamer-functionalized Au nanoparticles (NPs) are employed as reporters for surface-enhanced Raman scattering (SERS)-based cTnI detection. Only in the presence of cTnIs, the Au NPs are assembled onto the Au nanoplate and an NPs-on-nanoplate structure is formed. Scanning electron microscopy (SEM) images clearly show the cTnI-detected NPs-on-nanoplate architectures. Moreover, we quantitatively detected cTnIs by measuring SERS signals from the NPs-on-nanoplate structures. As a result, cTnIs in buffer solution are detectable at a low concentration of 100 aM (2.4 fg/mL), and cTnIs in human serum can be identified at a low concentration of 100 fM (2.4 pg/mL). Most importantly, the well-ordered aptamer on the Au nanoplates enabled us to diagnose nine AMI patients accurately. Based on these results, we insist that well-immobilized bioreceptors on Au nanoplates will be useful for the diagnosis of several diseases.Atomically flat Au nanoplates were synthesized by using a horizontal hot-wall furnace system with a quartz tube. The alumina boat containing an Au slug  was placed at the heating zone and the sapphire substrates were placed in the low-temperature zone. After the flowing of Ar gas for 30 min, the system was heated to 1100 °C and reacted for 60 min. The pressure of the system was kept as 10 Torr with an Ar gas flow rate of 100 sccm. After the reaction, atomically flat Au nanoplates could be obtained on the sapphire substrates.2, 50 mM Tris-HCl, pH 8.3) and distilled deionized water.As-synthesized Au nanoplates were transferred onto a Si wafer and incubated in a 1 µM probe aptamer solution , 10 mM NaCl, 5 mM KCl, 1 mM MgCl2, pH 7.4) at room temperature for 12 h. Next, the aptamer-immobilized nanoplates were rinsed with SELEX buffer  glycerol, 0.05% Tween 20, pH 8.0) or a hybridization buffer solution with 20% human serum (Sigma-Aldrich). Au NPs (~10 nm) were purchased from Sigma-Aldrich and used after centrifugation . Au NPs were dispersed in a final 500 µL of 1 µM reporter aptamer solution  at room temperature for 3 h. To remove excess reporter aptamers, the Au NP solution was centrifuged , the supernatant was removed, and resuspended in PBS (500 µL). For the detection of cTnIs, the prepared cTnI solution (1 mL) was added to the aptamer-immobilized Au nanoplates at 35 °C. After 6 h, the Au nanoplates were washed with the hybridization buffer and distilled deionized water. Next, the cTnI-captured Au nanoplates were incubated in the Au NP reporter solution at 35 °C for 30 min. Finally, the cTnI-detected Au NP-on-nanoplate structures were rinsed with deionized water and dried by N2 gas. The same experimental procedure was accomplished using troponin C , troponin T , immunoglobulin G , and avidin (Sigma-Aldrich) instead of cTnI for the selectivity test.The cTnIs were added to a hybridization buffer solution  or without (3 samples) AMI. The clinical study was approved by the Institutional Review Board (IRB) of the Kyung Hee University Hospital (KMC IRB 1104-04). Consent documents were obtained from all patients included in this study. All serum samples were stored at −80 °C until use. The concentration of cTnI in each of the clinical serum samples was determined using the SERS-based method and ELISA , respectively.SERS spectra were acquired using a homemade micro-Raman system based on an Olympus BX41 microscope . A He-Ne laser with 633 nm radiation  was used as the excitation source and the laser light was focused on the sample through a 50× objective  of the microscope. The spectra were measured with a thermodynamically cooled electron-multiplying charge-coupled device  mounted on the spectrometer with a 1200 groove/mm grating . A holographic notch filter was used to reject the laser light . The acquisition time was 1 min. SEM images were obtained from a Nova230 microscope (FEI Company). AFM images were obtained from a Park Systems XE-100 microscope.The atomically flat Au nanoplates were prepared by a vapor transport method . As-syntd value of the commercially available antibody against cTnI (20.8 nM) [20.8 nM) , the bin20.8 nM) ,5. The l20.8 nM) .−1, which corresponds to v(C=N)stretch, v(C-C)ring, v(C=C)ring, and v(C-N)stretch were clearly observed .In a typical hospital setting, after a blood separation process, serum is employed for the diagnosis of AMI. Therefore, cTnI-sensing methods for the diagnosis of AMI should operate effectively in serum. We examined the present method to detect cTnI in serum. −1 band intensities for several kinds of proteins . The concentrations of all proteins in the buffer were 10 pM. As shown in We further investigated the selective detection of cTnI using the aptamer-immobilized Au nanoplate platform. Finally, the aptamer-immobilized Au nanoplate platform was applied to the clinical diagnosis of AMI. Nine clinical samples  were collected from Kyung Hee University Medical Center and handled in accordance with approved IRB protocols at the hospital. The sex ratio of the samples was 4:5 , and the average age of the patients was 74.3, with a maximum of 87 and a minimum of 58. Although many biochemical parameters of heart-tissue origin have been used in the diagnosis of AMI from past to present, there is no consensus on the best cardiac biomarker . AnalysiWe report that an optimal aptamer-immobilized Au nanoplate platform enabled the sensitive and selective sensing of cTnI. The current study is important for the following reasons. First, the aptamer-immobilized Au nanoplate platform can detect cTnIs in the buffer at a low concentration of 100 aM (2.4 fg/mL). Compared to the previously reported cTnI sensing methods, the detection limit of this method is lower. Second, the high sensitivity of the aptamer-immobilized Au nanoplate platform can be maintained in serum tests. cTnIs in serum can be identified at a low concentration of 100 fM (2.4 pg/mL). This detection limit is much lower than the existing cutoff values, suggesting the potential diagnostic ability of the method. Third, the aptamer-immobilized Au nanoplate allowed us to diagnose nine clinical samples more accurately than conventional ELISA. In this regard, we expect that uniformly immobilized bioreceptors on Au nanoplates will be useful for the diagnosis of several diseases.


Background: Immunomodulatory properties of bisphosphonates (BP) are suggested to contribute to the development of medication-associated osteonecrosis of the jaw (MRONJ). Furthermore, bisphosphonate-derived immune modulation might contribute to the anti-metastatic effect observed in breast cancer patients. Macrophages are potential candidates for the mediation of immunomodulatory effects of bisphosphonates. The study aimed to investigate the influence of bisphosphonates alone and in combination with surgical trauma on systemic macrophage polarization (M1 vs. M2) using an in vivo rat model. Methods: A total of 120 animals were divided into four groups. Groups 2 and 4 were treated with 8 × 40 μg/kg body weight of the BP Zoledronate i.p. (week 0–7). Groups 3 and 4 were exposed to surgical trauma , whereas in Group 1 neither medication nor surgical trauma was applied. After 8, 10, 12 and 16 weeks, skin, lung and spleen were immunohistochemically examined for macrophage polarization via expression analysis of CD68, CD163 and iNOS using a tissue microarray (TMA). Results: A significant shift of macrophage polarization towards M1 was observed in skin, spleen and lung tissue of animals, with and without surgical trauma, treated with BP when compared to those without BP application. Surgical trauma did not cause a significant increase towards M1 polarization. Conclusions: BP application leads to a systemic pro-inflammatory situation in vivo, independent of surgical trauma, as evidenced by the shift in macrophage polarization towards M1 in various somatic tissues. This provides a possible explanation for the clinically observed anti-tumor effect of bisphosphonates and might also contribute to pathogenesis of MRONJ. Bisphosphonates (BP) such as Zoledronate are commonly prescribed antiresorptive drugs. They inhibit osteoclast function and thus prevent bone resorption ,2,3. TheBP-medication related osteonecrosis of the jaw (MRONJ) (BP) is one of the most severe complications of BP treatment. MRONJ is characterized by necrotic jaw bone that is exposed to the oral cavity due to a lack of epithelial coverage. It can lead to loss of teeth, infections, jaw fractures and eventually to the loss of large parts of the jaw, requiring extensive surgical reconstruction ,6. The pThe immunological microenvironment of the affected tissues and, in this context, the polarization of macrophages (M1 vs. M2) is involved in the pathogenesis of inflammatory and malignant diseases. M1-polarized macrophages act as pro-inflammatory and are associated with tissue destruction but also with tumor defense ,11. In cIn human jaw bone samples affected by BRONJ (BP), a significant increase of macrophage infiltration as well as M1 macrophage polarization was demonstrated . These dA potential systemic shift of macrophage polarization in BP-treated individuals might explain the anti-metastatic effect of BP as well as the occurrence of MRONJ. It is well-accepted that the cell density and polarization of macrophages contribute to the progression of several malignancies . In thisThe effects of BP treatment can be examined not only by studying the place of surgery and at the locally induced defect. The spleen is a secondary immune organ. It is involved in the regulation of immune responses locally and in the whole body. Additionally, it is of importance for inflammatory and degenerative diseases and modulates the innate and adaptive immunity. Spleen, lung and skin are populated with cells of the macrophage system. Therefore, BP treatment and/or a distant surgical trauma can also lead to a shift towards pro-inflammatory M1 polarization of macrophages in these organs.In a Wistar rat model, the effect of the bisphosphonate Zoledronate on infiltration and polarization (M1 vs. M2) of tissue macrophages in different organs  was investigated in this study. It was tested if Zoledronate caused a systemic shift of macrophage polarization towards M1. Additionally, the contribution of surgical trauma to bisphosphonate-mediated alterations in macrophage infiltration and polarization was analyzed.For this analysis, the samples of 108 animals were available. The drop-outs were caused by animals that had to be sacrificed because abort criteria  occurred prior to tissue harvesting.The macrophage markers analyzed in this study showed a staining of the plasma membrane and the cytoplasm that was analogue to previously published macrophage analyses in human cancer and bone specimens ,27. Exemp = 0.004; p = 0.017)  , the CD68 labeling index in the subepithelium of the skin tissues (dermis and subcutaneous tissue) in bisphosphonate (BP)-treated animals was significantly increased compared to untreated controls  . In cont= 0.017) . A simil< 0.001) b. There < 0.001) c. Consid< 0.001) .p = 0.007) (p = 0.012) (p < 0.001)  . Accordi= 0.012) . Conside= 0.012) d,e; howe< 0.001) f. ExpresIn the epithelial compartment of the skin, low levels of macrophage infiltration were detected. The epithelial macrophage expression is provided in the p = 0.001)  application compared to controls without antiresorptive medication . This re= 0.001) b. In con= 0.001) a,c.p = 0.005) (p = 0.027) (p < 0.001)  in lung tissue was significantly higher in BP-treated animals compared to untreated controls  (= 0.005) d. Accord= 0.027) e. The iN< 0.001) e. Similap = 0.006)  application compared to untreated animals of all subgroups  . In case= 0.006) c. In con= 0.006) a,b.p = 0.03) (p = 0.008) or only cases with surgical trauma (p = 0.166) (p = 0.02) (p = 0.002)  in BP-treated animals compared to rats with no treatment  ( = 0.03) d. Simila= 0.166) . The CD1 = 0.02) e. Accord = 0.02) . In the = 0.002) f.The white pulp of the spleen showed a significantly lower infiltration of macrophages compared to the red pulp . Low iNOS and CD68 along with an extremely low CD163 expression were observed in the white pulp compared to the red pulp . However, considering only BP-treated animals, a significantly decreased iNOS labeling index as well as iNOS/C68 and iNOS/CD163 expression ratio in rats with surgical trauma compared to rats without surgery was seen see .p = 0.001)  b. The hi= 0.001) . In cont= 0.001) . Additio= 0.001) . The dif= 0.001) . The hig= 0.001) .p = 0.031)  .The current study revealed that application of the bisphosphonate (BP) Zoledronate leads to a systemic shift of the polarization of tissue macrophages towards M1. The M1-shift was observed in the skin, spleen and lung of the BP-treated Wistar rats when compared to untreated controls. The BP-induced M1 shift was noted in cases without and with surgical trauma. This could explain the anti-metastatic effect of BP and contribute to the pathogenesis of MRONJ.In MRONJ, wound healing is impaired . MacrophIt is believed that the initially infiltrating M1 macrophages repolarize towards M2 during healing processes . The M2 The anti-metastatic effect of BP even in cases with an absence of bone metastases must be mediated by BP-derived action that goes beyond the inhibition of osteoclastic bone resorption . SeveralIn breast cancer, macrophages can contribute to up to 50% of total cell mass and are the main players in tumor immunology . A conneThere are two main lineages of macrophages described: firstly, tissue-resident macrophages that originate from embryonic precursors and have the potential of self-renewal, and secondly, bone-marrow-derived macrophages that infiltrate from the blood stream . The tisreg). This is mediated via the M2 cytokine TGF-beta [reg cells act immunosuppressive and are considered as an important tumor-promoting cell type [reg generation through reduced M2 polarization in the red pulp could also contribute to BP-derived anti-metastatic immune modulation.It was shown that spleen red pulp macrophages are capable of inducing regulatory T-cells . NO is an important pro-inflammatory mediator with an immense effect on the immune system and different immune cells. Thus, the missing correlation between CD68 and iNOS could be explained by the fact that iNOS is produced by a variety of cells after induction by cytokines or other stimuli. Indeed, many studies have shown that iNOS is expressed by cells other than macrophages such as activated T cells and mature dendritic cells (mDCs) . Therefon = 30) included animals which were not untreated and served as control. For 8 weeks, the animals of Groups 2 (n = 32) and 4 (n = 20) were treated with the amino-bisphosphonate Zoledronate  at a dose of 40 μg/kg body weight i.p. one time per week (n = 26) and 4 (n = 20) were subjected to surgical treatment that consisted of extraction of the mandibular first molar on one side of the jaw and the osteotomy of a unilateral tibia bone followed by an immediate rigid osteosynthesis with an 8-hole 2.0 mini plate and 4 screws  after 8 weeks  at a thickness of 2 µm, and Hematoxylin and Eosin (H&E) staining was performed. All stained slides were scanned and digitized with the method of “whole slide imaging” in cooperation with the Institute of Pathology of the University of Erlangen-Nürnberg using a Pannoramic 250 Flash III Scanner . The digital slides were analyzed using the software Case Viewer (3DHistech). For establishing the tissue micro array, representative punch locations were annotated to each digital slide. For skin, the epithelia were placed in the middle and three punches of 1 mm diameter were taken from each sample . For splThe staining procedure was performed using the LSAB2 K0609 kit  and an autostainer . The following primary antibodies were applied: anti-CD68 1:50 , anti-CD163 1:100  and anti-iNOS 1:100 . All TMA slides were scanned and digitized with the method of “whole slide imaging” in cooperation with the Institute of Pathology of the University of Erlangen-Nürnberg using a Pannoramic 250 Flash III Scanner  as previously described ,27.All TMA slides were imported into the digital pathology image analysis software Tissue Studio . This software analyzed the digital slides and detected automatically the TMA cores and the tissue . After tFor the different tissue types and the various stains, it was necessary to adjust the parameters according to the intensity of the stains. The parameter settings were identical for the tissue types and stains. Representative results are shown in 2 (25 and 25) and the typical cell size in µm2 (450 and 350) were set. Because of the similarity of the spleen anti-CD68 staining to a nuclear marker staining, the nuclear marker analysis tool of Tissue Studio was used for counting of CD68-expressing cells. The parameters hematoxylin threshold (0.05), Immunhistochemical (IHC) threshold (0.55) and typical nucleus size in µm2 (52) were set. The spleen tissue was divided into these ROI: white pulp and red pulp.Assessment of spleen samples with anti-CD163 and anti-iNOS was performed using the cytoplasmatic analysis tool of Tissue Studio. The parameters hematoxylin threshold (0.07 and 0.08), marker threshold (0.35 and 0.3), typical nucleus size in µm2  were set. For the skin tissue  the nuclear marker tool was used and the parameters hematoxylin threshold , IHC threshold  and typical nucleus size in µm2  were set. For analysis, the skin tissue was divided into epithelium (epidermis) and sub-epithelium compartment. The sub-epithelium contains the dermis and the subcutaneous tissue.For lung tissue with anti-CD68, anti-CD163 and anti-iNOS staining, the nuclear marker analysis tool of Tissue Studio was used and the parameters hematoxylin threshold , IHC threshold  and typical nucleus size in µmAll parameters were set to ensure that the positive and negative cells shown in the Tissue Studio software preview corresponded approximately to human evaluation. Additionally, it was ensured that total cell count within a tissue type was approximately equal in different stainings for better comparability.2) for each core and ROI. Additionally, it contained screenshots of each core in original, with colored markings of positive and negative cells and with colored markings of the division into the different ROI.The Tissue Studio software calculated the number of positive and negative cells, and the tissue area per core and per ROI. For each tissue stain, the Tissue Studio software was run with the adjusted settings and the results were saved in a folder which contained a CSV file with all the positive and negative cells per core and ROI, and contained a CSV file with the area information  file. Therefore, a program in the programming language Python was written. Using the Python-based program, the cell and area information of all of the individual CSV result files from the Tissue Studio software were collected and data were summarized in one Excel table for the statistical analysis. Also, the ratios of the different stains  were calculated and the data were added to the table.2 was determined as the number of positive cells per mm2 within the tissue. The labeling index was calculated dividing the number of positive cells by the number of all cells (positive + negative). The data were investigated for a significant relationship between bisphosphonate application, surgical trauma and a redistribution of macrophage markers. The results were expressed as the median values. Also, standard deviation (SD) was determined. Box plot diagrams represent the median, the interquartile range, minimum (Min) and maximum (Max). The statistical analyses were performed with the software SPSS 22 for Mac OS  using the Mann-Whitney-U-Test. Two-sided, adjusted p-values ≤ 0.05 were considered to be significant.To analyze the immunohistochemical staining, the cell count per mmTo our knowledge, this is the first study performing a systematic analysis of the polarization of tissue macrophages in a bisphosphonate animal model. We can show a systemic shift of macrophage polarization towards M1 in bisphosphonate-treated animals. This was present in tissues with a dominating population of tissue-resident macrophages and in organs with predominantly bone-marrow-derived macrophages. The BP-derived shift from M2 towards M1 could contribute to the explanation of impaired wound-healing in BP-affected soft tissue and might also explain the clinically observed anti-metastatic effect of BP.


Cisplatin [cis-diamminedichloroplatinum II] is an extensively prescribed drug in cancer chemotherapy; it is also useful for the treatment of diverse types of malignancies. Conversely, cisplatin is associated with a range of side effects such as nephrotoxicity, hepatotoxicity, gastrointestinal toxicity, and so on. Myricetin -4chromenone) is a very common natural flavonoid found in fruits, tea, and plants. It has been found to have high-value pharmacological properties and strong health benefits. To examine the role of myricetin in colon toxicity induced by cisplatin, we conducted a concurrent prophylactic study in experimental animals that were treated orally with myricetin for 14 days at two doses—25 and 50 mg/kg of body weight. On the 14th day, a single intraperitoneal injection of cisplatin (7.5 mg/kg body weight) was administered in all groups except control. The effects of myricetin in cisplatin-induced toxicity in the colon were assessed in terms of antioxidant status, phase-II detoxification enzymes, the level of inflammatory markers, and goblet cell disintegration. Myricetin was found to restore the level of all the antioxidant enzymes analyzed in the study. In addition, the compound ameliorated cisplatin-induced lipid peroxidation, increase in xanthine oxidase activity, and phase-II detoxifying enzyme activity. Myricetin also attenuated deteriorative effects induced by cisplatin by regulating the level of molecular markers of inflammation , restoring Nrf-2 levels, and controlling goblet cell disintegration. The current study reinforces the conclusion that myricetin exerts protection in colon toxicity via up-regulation of inflammatory markers, improving anti-oxidant status, and protecting tissue damage. Cisplatin [cis-diamminedichloroplatinum (II) or cisplatium] is the first generation of anti-cancer drug that possesses the platinum coordination complex with a square planar geometry. Cisplatin is a solid white crystalline powder that is stable at ambient temperature . It was Many reports support the notion that cisplatin is not specific in action at the site of cancers, but affects rapidly dividing cells elsewhere in the body, e.g., the epithelial cells of intestines. It does so by massively producing free radicals and generating oxidative and nitrosative stress ,6,11,12.Myrica nagi in 1896. Later, its structural configuration was observed by the same authors  (DTNB), thiobarbituric acid (TBA), folin ciocalteau reagent (FCR), nicotinamide adenine dinucleotide phosphate oxidized (NADP), flavin adenine dinucleotide (FAD) and myricetin were purchased from Sigma . Sodium dihydrogen phosphate, sodium potassium tartrate, pyrogallol, sodium hydroxide, and di-sodium hydrogen phosphate were obtained from E. Merck Limited, India. Cisplatin was procured from Dr. Reddy’s Laboratories, India.To study the cisplatin-induced oxidative burst and inflammatory response in the colon, a prophylactic treatment with myricetin was conducted. In this study, 4 groups with 6 Wistar male rats in each group were arbitrarily allocated. Myricetin was suspended in 5% sodium carboxymethyl cellulose (CMC-Na) . The firg with temperature set at 4 °C for the separation of nuclear debris. The supernatant obtained was centrifuged for 20 min at 15,520× g at 4 °C to obtain Post-mitochondrial Supernatant (PMS), which was also used as the source of various enzymes.Colon tissues free of any extraneous material were detached rapidly from the euthanized rats and were perfused in ice-cold saline (0.85% sodium chloride), then homogenized by means of in ice-cold phosphate buffer . After filtering the homogenate through a muslin cloth, it was centrifuged for 10 min at 1744× l-lysine finished glass slides. The tissues were de-waxed in xylene and rehydrated through a graded sequence of water and ethanol. Slides were then stained for 30 min in Alcian blue (1%: pH 2.5) with 3% acetic acid solution. To remove non-specific staining, the glass slides were rinsed in 3% acetic acid for 60 s after staining and then washed with distilled water. The tissue sections were then counterstained using neutral red (0.5% aqueous solution) for 20 s and then dehydrated in ethyl alcohol. The sections were then covered using mounting media and the slides were examined under the microscope.Formalin-fixed and paraffin-embedded colonic sections of size 4 μm from tissue blocks were mounted on poly 2O2 in methanol for 10 min and then were subjected to power block for 10 min to block non-specific binding. The sections were rinsed in TBS and incubated at 4 °C overnight inside a humidified chamber with the primary antibody, then washed in TBS. The tissue sections were incubatedwith biotinylated goat anti-polyvalent secondary antibody for 20 min and rinsed in TBS. The sections were incubated again with streptavidin peroxidase plus for 30 min. The sections were washed in TBS and developed with 3,3′-Diaminobenzidine (DAB) solution until they became brown. The sections were counterstained with Mayer’s hematoxylin and then mounted using the mounting media. Finally, slides were evaluated under the microscope. Primary antibodies were rabbit anti-Nrf-2 (dilution 1:200), and rabbit NF-κB (dilution 1:100).Sections of 4 μm each from the tissue blocks which were formalin-fixed and paraffin-embedded were mounted on poly L-lysine coated glass slides. The sections, after dewaxing them in xylene and then rehydrated through graded series of ethanol and water, went through a process of antigen retrieval in sodium citrate buffer ; after that, slides were cooled for 15 min. Then they were washed with tris-buffered saline (TBS) 3 times each for 5 min. To reduce the activity of endogenous peroxidase, the slides were incubated in 3% Hg for 10 min. Optical density was measured at 535 nm to assess the quantity of malondialdehyde (MDA) formed. The units of the results obtained were for nmol TBA formed/h per g tissue at 37 °C  were incubated together for 10 min in the dark at room temperature. Nitrites were measured at 540 nm with spectrophotometer. The level of nitrites was derived by drawing a standard curve of NaNO2 .The neutrophil quantification is measured as a level of myloperoxidase (MPO) activity and was carried using the Bradley et al. method (1982) .2O2) was implemented [The quinone reductase enzyme activity was assayed by the method of Benson et al. (1980) . Marklunlemented . The actlemented . The actlemented . The glulemented . The redlemented . GST actlemented . The enzlemented .G6PD activity was assayed by the method of Zaheer et al. (1965) . TNF-α lp < 0.05 for all comparisons.The data from the individual groups are presented as the mean ± standard error of the mean (SEM). The differences between the groups were analyzed using analysis of variance (ANOVA), followed by Tukey-Kramer multiple comparisons test. The minimum criterion for statistical significance was set at All procedures for using experimental animals were checked and proper permission was obtained from the “Institutional Animal Ethical Committee (IAEC)”  that is fully accredited by the Committee for Purpose of Control and Supervision on Experiments on Animals (CPCSEA), New Delhi, India.p < 0.001) in cisplatin-induced group II as compared to group I. Myricetin pre-treatment noticeably decreased the level of MDA in group III (p < 0.05) and group IV (p < 0.001), respectively, when compared to group II (p < 0.001) in the MPO activity in group II when compared to control group I. Myricetin for both the doses in group III (p < 0.05) and group IV (p < 0.01) was instrumental in bringing back the unusual levels of MPO to normal (p < 0.001) was observed in nitrite levels in group II when compared to control group I. Myricetin for both the doses (25 and 50 mg/kg body weight) in group III (p < 0.01) and group IV (p < 0.001) was effective in normalizing nitrite levels  as compared to control group I. Myricetin pre-treatment revealed a visible increase in reduced glutathione level in group III (p < 0.05) and group IV (p < 0.05) when compared with group II. Cisplatin administration caused a decrease in the activities of GPx (p < 0.001), GST (p < 0.05), G6PD (p < 0.001) and GR (p < 0.001) in group II as compared to group I. Myricetin supplementation for both doses (25 and 50 mg/kg body weight) showed remarkable improvement in restoring levels of all the above-mentioned enzymes. Myricetin at higher dose showed higher potential (p < 0.001) in restoring the glutathione-dependent enzyme activity (Reduced glutathione levels were depleted in cisplatin-treated group II (activity .p < 0.001) in group II when compared to group I. In addition, the activity of superoxide dismutase (SOD) increased significantly (p < 0.001) after cisplatin treatment in group II as compared to group I. Treatment with myricetin at a dose of 25 mg/kg body weight augmented the activities of QR (p < 0.05) and catalase (p < 0.05) in group III as compared to group II, while the activity of SOD was markedly lower (p < 0.05) in group III as compared to group II. The higher dose of myricetin (50 mg/kg b. wt.) also showed a significant increase in the activities of catalase (p < 0.001) and QR (p < 0.01) in group IV as compared to group II, while the activity of SOD decreased (p < 0.001) in group IV as compared to group II (The activities of quinone reductase (QR) and catalase were drastically lowered (group II .p < 0.001) in group II as compared to group I. Myricetin treatment markedly lowered the level of XO at both the doses in group III (p < 0.01) and group IV (p < 0.01), as shown in The activity of XO was significantly enhanced  as compared to the cisplatin-treated group II. Pre-treatment with myricetin markedly inhibits the production of TNF-α in the groups III and IV when compared to the cisplatin-treated group II (p < 0.001) as compared to cisplatin-treated group II. Pre-treatment with myricetin markedly inhibited the production of IL-6 in groups III (p < 0.01) and IV (p < 0.001) when compared to the cisplatin-treated group II (Quantitative production of TNF-α was assessed in colon via cisplatin treatment. We observed that there was a marked difference in the level of pro-inflammatory cytokines in control group I (group II a. The efgroup II b.Colonic sections treated with cisplatin in group II showed a massive disintegration of the goblet cells compared to the control group I. Myricetin supplementation at both the doses (25 and 50 mg/kg body weight) prevented the disintegration of the goblet cells and prevented colonic damage .The current study was undertaken to analyze the protective effects of myricetin in cisplatin-induced colon toxicity in an experimental model. Cisplatin-induced colon toxicity is extensively documented in the literature ,11. Emes2O2), which play a crucial role in the instigation of lipid peroxidation [Many mechanisms have been put in place to explain the actual phenomenon involved in cisplatin toxicity in colon/intestines, but the most accepted one involves reactive oxygen species (ROS) generated by cisplatin ,57. Accuxidation . Chang exidation  reportedxidation . One of xidation ,11,60,62xidation  Figure O2, whichEnzymatic and non-enzymatic antioxidants act as the most important defense to counter free radicals in any biological system ,65. GlutAs reported earlier by many groups, and as discussed above, cisplatin-induced toxicity was closely associated with the generation of ROS ,65. CispInflammation and oxidative insult are together considered crucial factors responsible for cisplatin-induced intestinal toxicity. Nuclear factor erythroid 2-related factor 2 (Nrf-2) has been shown to up-regulate the expression of the various antioxidants while role of ROS in inflammation in NF-kB mediated inflammation has been studied in cisplatin-induced organ injury . NuclearCisplatin exposure is reported to be linked with increased levels of neutrophils and macrophages ,87. ThesThe pro-inflammatory cytokines (IL-6 and TNF-α) have been shown to play a crucial role in cisplatin-induced organ injury ,75. TNF-In the colon, mucosal barriers have multi-pronged defense mechanisms that specifically respond to oxidative insults in order to maintain constant homeostasis . Many prThe actual mechanism of action of myricetin in preventing cisplatin toxicity has not been revealed yet. The findings from the current study show that myricetin demonstrated a protective effect in cisplatin-induced toxicity by controlling oxidative stress, tissue damage and inflammation. A combination of myricetin and cisplatin as combinational therapy offers a better alternative for reducing the side effects of cisplatin and improving chemotherapeutic value. Further pre-clinical and clinical studies should be undertaken for the above combinations to determine the actual mechanisms and effects in humans.


Previous studies demonstrated that hyperglycemic glucose concentrations are observed in children that are overweight or have obesity. The aim of this study was to evaluate the effect of a 12 month lifestyle intervention on free-living glycemic profiles in children that were overweight or had obesity, and the association of the alterations with changes in cardiovascular risk parameters. BMI z-score, free-living glycemic profiles, continuous overlapping net glycemic action (CONGA), and cardiovascular parameters were evaluated before and after a multidisciplinary lifestyle intervention, in 33 non-diabetic children that were overweight or had obesity. In children with a decrease in BMI z-score, the duration which glucose concentrations were above the high-normal threshold (6.7 mmol/L) and the glycemic variability decreased significantly. In these children, a decrease in median sensor glucose was associated with decreases in LDL-cholesterol, and systolic and diastolic blood pressure z-score. A decrease in BMI z-score was associated with a decrease in CONGA1, 2, and 4. In conclusion, the glycemic profiles in free-living conditions in children that were overweight improved in children with a decrease in BMI z-score after lifestyle intervention. In those children, changes in median sensor glucose concentrations were associated with changes in LDL-cholesterol and blood pressure z-scores. These results suggest that glucose homeostasis can improve after one year of lifestyle intervention and that these improvements are associated with improvements in cardiovascular health parameters. It is well acknowledged that children that are overweight or have (morbid) obesity are at risk for developing type 2 diabetes mellitus (T2DM) and cardiovascular diseases (CVD) ,2. ThereIn a recent study, we demonstrated that, besides the presence of an increased CVD risk, glucose homeostasis is already disturbed in these children . HypoglyIn adults with T2DM, glucose disturbances were shown to be reversible, since lifestyle interventions improving dietary behavior or physical activity resulted in a significant improvement of glycemic variability in free-living conditions ,17,18,19This study was designed and conducted within the setting of the Centre for Overweight Adolescent and Children’s Healthcare (COACH) at the Maastricht University Medical Centre (MUMC+). Within COACH, the health status of children with overweight and with (morbid) obesity and their families was evaluated; they were monitored and received lifestyle coaching as described previously . BrieflyFurthermore, participation in sports activities in groups and activities aimed at increasing nutritional knowledge were offered. A follow-up assessment including all of the examinations performed during the initial assessment was offered annually to all children.n = 10). Finally, 33 children were eligible for inclusion. Informed consent was obtained from all subjects and/or their parents. The study was conducted according to the guidelines administered by the Declaration of Helsinki and approved by the medical ethical committee of the MUMC+. It is registered at ClinicalTrial.gov as NCT02091544.All 43 children with complete CGM sensor data at baseline and who had additional CGM sensor measurements after 12 months of intervention were considered for inclusion in this study. Children with incomplete CGM sensor data after 12 months of intervention were excluded from this study (2 in adults), having obesity (comparable to a BMI of 30 kg/m2 in adults), or having morbid obesity (comparable to a BMI of 35 kg/m2 in adults) [Anthropometric measurements were acquired while children were barefoot and wearing only underwear. Body weight was determined using a digital scale (Seca), and body length was measured using a digital stadiometer . Body mass index (BMI) was calculated and BMI z-scores were obtained using a growth analyzer , based upon reference charts of the Dutch nationwide growth study . Based o adults) . Waist c adults) . Ethnici adults) .Fasting plasma glucose concentrations , serum insulin concentrations , and HbA1c concentrations  were determined. After obtaining the fasting blood sample, an OGTT was performed. An amount of 1.75 grams of glucose per kilogram of bodyweight was dissolved into 200 mL water, with a maximum of 75 grams of glucose in total, and given orally. Plasma blood glucose concentrations were measured every thirty minutes over two hours. Impaired fasting glucose , IGT (≥7.8 glucose <11.1 mmol/L after 2 hours), T2DM (fasting glucose ≥7.0 mmol/L or glucose ≥11.1 mmol/L after 2 hours), and elevated HbA1c concentrations (≥5.7%) were defined according to the American Diabetes Association (ADA) criteria . In thisGlucose concentrations in free-living conditions were measured using a CGM sensor , as described previously . In shorz-scores were calculated according to reference values based on height and gender [Fasting lipid and lipoprotein profiles, including serum total cholesterol (TC), serum LDL-cholesterol (LDL-C), serum HDL-cholesterol (HDL-C), and serum triglyceride concentrations, were measured . Daytime blood pressure (BP) was measured during a period of 1.5 hours—approximately 20 times, with an interval of three minutes between each measurement—using the Mobil-O-Graph (I.E.M. GmbH). Based on these 20 measurements, the mean BP was calculated. The size of the cuff depended on the circumference of the upper arm. Systolic and diastolic BP d gender .z-score, sensor glucose measurements, and cardiometabolic risk parameters at baseline and after 12 months of lifestyle intervention were compared using the paired Student’s t-test, the Wilcoxon signed-rank test, or the χ2 test, as appropriate. Correlations between variables were determined by Spearman’s correlation analysis. Data are presented as means with standard deviations or as medians with the minima and maxima. For all analyses, a p-value below 0.05 was considered to be statistically significant.All statistical analyses were performed using SPSS 20.0 for Windows . BMI n = 3), 42% (n = 14) were obese, and 49% (n = 16) were morbidly obese. Despite the wide range in BMI z-scores, all children had fasting plasma glucose concentrations within the normal range (<5.6 mmol/L). The mean HOMA-IR was 3.31 ± 1.61. Based on these HOMA-IR values, insulin resistance was present in 58% (n = 19) of the children. One child was classified as having impaired glucose tolerance. HbA1c concentrations were elevated in 27% (n = 9) of the children. After 12 months of lifestyle intervention, the BMI z-score did not improve significantly in the complete group (p = 0.206), whereas there was a significant improvement in weight status classification (p < 0.001), i.e., a shift from morbidly obese to obese, or obese to overweight (p = 0.018), LDL-C (p = 0.025), and HbA1c concentrations (p < 0.001) decreased significantly in the whole group after 12 months of lifestyle intervention (p = 0.014) and HOMA-IR (p = 0.014) increased significantly. The characteristics of the children at baseline and after 12 months of lifestyle intervention are presented in Thirty-three, predominantly Caucasian (85%) children  with a mean age of 12.5 ± 3.2 years were included. At baseline, 9% were overweight  children with a decrease in BMI z-score and (2) children with an increase in BMI z-score. Sixty-one percent (n = 20) of the children successfully improved their BMI z-score, with a significant decrease of −0.24 ± 0.15 units (p < 0.001). BMI z-score increased significantly by 0.21 ± 0.17 units (p = 0.001) in the remaining 39% (n = 13) of the children. Children with a decrease in BMI z-score over time were significantly younger at baseline as compared with children that showed an increase in BMI z-score. There were no significant differences at baseline between these groups regarding anthropometric measurements and cardiovascular risk parameters (z-score. HbA1c concentrations improved significantly in both groups (z-score (Based on changes in BMI rameters . Signifih groups . A signi(z-score .p = 0.431) (p = 0.048) . The median sensor glucose concentration at baseline was 5.0 (3.2–7.3) mmol/L and did not change significantly after 12 months of lifestyle intervention (= 0.431) . CONGA1 = 0.048) , but CONn = 21) of the children reached high-normal sensor glucose concentrations, defined as values ≥6.7 mmol/L, during the 48-hour measuring period. Of these children, six out of 21 no longer reached these high-normal sensor glucose concentrations after 12 months of lifestyle intervention. In addition, a significant decrease in the duration of glucose concentrations being ≥6.7 mmol/L (p = 0.001) was demonstrated after 12 months of intervention in the complete group  of the children showed sensor glucose concentrations ≥7.8 mmol/L during the 48-hour measuring period. The duration of glucose concentrations exceeding this threshold did not change significantly after 12 months of lifestyle intervention (p = 0.408) (n = 2) at baseline, all sensor glucose concentrations were <6.7 mmol/L after 12 months of intervention. Furthermore, changes in the opposite direction were also observed, i.e., eight out of 12 children with glucose concentrations <6.7 mmol/L at baseline exceeded this threshold after 12 months of lifestyle intervention. Four of these children did not show a decrease in BMI z-score after the intervention. At baseline, 36% (= 0.408) . Howevern = 24) of the children reached sensor glucose concentrations below 3.9 mmol/L at baseline. The total duration for hypoglycemic sensor glucose concentrations did not change significantly after 12 months of intervention (p = 0.323)  .z-score, the duration of sensor glucose concentrations being ≥6.7 mmol/L decreased significantly (p = 0.009), and in the children who demonstrated an increase in BMI z score, there was a trend towards a shorter duration of sensor glucose concentrations ≥6.7 mmol/L (p = 0.060) , whereas there were no significant changes found in children with an increase in BMI z-score  (z-score.In the children who demonstrated a decrease in BMI = 0.060) . CONGA1 ctively) . Sensor z-score  and delta DBP z-score  in the whole group. The delta of the median sensor glucose concentration was not associated with the delta of the anthropometric measurements or the other cardiovascular risk parameters. Delta CONGA1, delta CONGA2, delta CONGA4, and delta CV were not associated with alterations in any of the anthropometric measurements or cardiovascular risk parameters in the whole group.After 12 months of lifestyle intervention, the delta of the median sensor glucose concentration showed a positive association with the delta SBP z-score, there was a positive association between the delta BMI z-score and delta CONGA1 , delta CONGA2 , delta CONGA4 , and delta CV . Moreover, in these children, the delta median sensor glucose was positively associated with the delta LDL-C , delta SBP z-score , and delta DBP z-score . The delta CV was positively associated with the delta TC  and delta serum triglyceride concentration . In children with an increase in BMI z-score, no associations were found between the delta BMI z-score and alterations in sensor glucose measurements. In these children, the delta maximum sensor glucose concentration showed inverse associations with delta TC  and delta LDL-C . Correlation coefficients stratified for changes in BMI z-score are presented in z-score.In the children with a decrease in BMI z-scores. These associations were only present in children with a decrease in BMI z-score. Our results suggest that an on-going, tailored, outpatient lifestyle intervention can result in the improvement of glycemic profiles in free-living conditions and that these improvements may coincide with a decreased CVD risk in children with overweight and with (morbid) obesity.This is the first study investigating the effect of a long-term lifestyle intervention on glycemic profiles in free-living conditions in children with overweight and with (morbid) obesity. Our results demonstrate that the durations for which glucose concentrations were in the high-normal range and the glycemic variability calculated as CONGA1 decreased significantly after 12 months of lifestyle intervention. Furthermore, the delta of the median glucose concentrations in free-living conditions was positively associated with the deltas of the SBP and DBP z-score [z-score coincides with improvements of glycemic profiles. The duration of glucose concentrations being in the high-normal range and the CONGA1 only improved in children with a decrease in BMI z-score. Notably, associations between the delta of the median sensor glucose concentrations and deltas of the SBP and DBP z-scores were only found in the subgroup of children with a decreased BMI z-score. It is tempting to suggest that a decrease in BMI z-score is the result of lifestyle improvements. Dietary composition and quality as well as physical activity were important aspects of the lifestyle intervention and are factors well-known to interact with glucose homeostasis [z-score. In a previous cross-sectional study in children with overweight and with (morbid) obesity, we demonstrated that glycemic profiles in free-living conditions were aberrant . This waz-score . We now eostasis . As menteostasis ,17,18,19z-score, HbA1c concentrations improved significantly after 12 months of lifestyle intervention. By contrast, we found a significant increase in fasting insulin levels in the children with an increase in BMI z-score after the intervention. These findings are in line with a previous study in children, in which there was a strong association between an increasing degree of insulin resistance and increasing adiposity [z-score, lifestyle changes made during the intervention might have positively affected HbA1c concentrations despite an increase in BMI z-score.In the current study, glycemic variability was assessed using the CONGA and the coefficient of variation. In general, high glucose variability is thought to be harmful for peripheral vascular function ,12,13,14diposity . Similardiposity . These fdiposity ,37. Takiz-score. Furthermore, it was shown that the delta of the median glucose concentration was associated with the deltas of the SBP and DBP z-scores, only in children with a decrease in BMI z-score. Interestingly, these correlations between the delta of the median glucose concentrations and deltas of SBP and DBP z-scores were not found for the deltas of the CV and CONGA1, CONGA2, or CONGA4. In children with an increase in BMI z-score, HbA1c concentrations improved significantly, while cardiovascular risk parameters showed no significant improvements. These results suggest that changes in glucose homeostasis may, but do not necessarily, coincide with changes in cardiovascular risk parameters.The exact underlying mechanisms and sequence of events resulting in glucose dysregulation are not fully understood, but there is strong evidence suggesting a link between glucose dysregulation and dyslipidemia . In thisDue to the long-term follow-up of our intervention, we did not include a control group with random assignment of treatment, because it was not ethically justifiable to keep children in a control program for a prolonged period of time and withhold treatment from them. This can be considered as a limitation of this study. Furthermore, the cohort size of our study might seem small, and therefore the affirmation of our findings in larger cohort studies is certainly recommendable. However, considering the current literature and the novelty of investigating sensor glucose measurements in children without a diagnosis of T2DM, it can be argued that we have a relevant study population size and that the results of this study might create awareness that further research is needed. It would have been valuable if healthy children with a normal weight were included in this study as a reference population for the normality of glycemic profiles, since the current evidence in this population is limited ,39. Addiz-score, glycemic profiles in free-living conditions improve after 12 months of lifestyle intervention, as demonstrated by a decrease in the duration of time that glucose concentrations are in the high-normal range and by the decrease in CONGA1. Changes in median glucose concentrations are associated with changes in SBP and DBP z-scores in children with overweight and with (morbid) obesity, but only in those who showed a decrease in BMI z-score. These results suggest that a lifestyle intervention can result in the improvement of glucose homeostasis and that these improvements are also associated with improvements in cardiovascular health parameters. Next, long-term follow-up studies are necessary to evaluate whether the improvement of glycemic profiles in free-living conditions during childhood results in long-term health benefits.In children with a decrease in BMI